Hi Fahar

I think you have to be sure that adding the media to the cell line should be always in the safety cabinet after it  being cleaned up with ethanol spread over the surface, also make sure you spray every things that going to be in the cabinet before locating them in there, your hands etc.. I am not sure whether the pipettes is causing the contamination or not due to reusing them.

hope this would be helpful

good luck

Hi Fahar,

I am wondering what kind of contamination you have? Bacterial or fungal?

From my experience, the bacterial contamination always come from the the sterile technique, so it will help if you spray everything, including your hand, with 70% ethanol before put things in the safety cabinet or culture hood, as the previous comment recommended. Do you include antifungal-antibacterial in your media? That will decrease the chance of contamination too, but it depends on the limitation of your culture.

If you suspected that the source is your media, you can refilter it. Other sources can be the water bath that has been used to warm up media, I recommend adding Roccal in the water.

I have reused pipette tips as well. I always make sure that the tip will not be touched before reuse it and my culture is fine.

Hope this help.

Good luck!

Hi Fahar,

If you suspect that contamination comes from the pipettes, I recommend you the use of tips with filter. Although these pipette tips are more expensive, in the end you will save money and time. In our lab we have been using these tips over the last 5 years and we only had 2-3 contaminations.

Good luck!

I think better to use use of tips with filter.Don,t mix your Pipettes.

I personally would not reuse normal tips for cell culture, it's too risky. Filter tips are a must to help prevent contamination, especially if media is being shared.

That being said, I have experienced a contamination issue identical to what you're describing (and we were using sterile filter tips, fresh media, etc.)! In our case, what actually ended up doing the trick was sanitizing and then autoclaving the shelves & water pan within our incubator - just an idea to try if the other modifications don't end up fixing your issue.

At least for cell culture do not reuse the tips, pipettes after autoclaving.  Avoid this.  Use disposable supplies.   you will save time and money in long run.  Check your media is contaminated before using them.

Never re-use anything in cell culturing. Use disposables!

It seems like there is an issue about reusing pipette tip here, so I am feeling like I need to clarify ‘reusing pipette tip’ so that no one else confuses with this and for the benefit of others that have a similar problem.

As cell culture is the delicate system that requires extended sterile technique and autoclaved clean, I am bound to do that too. Everything used in my cell culture is disposable, however, let me clarify the word reuse pipette tip.

Let’s say when I change culture media for 48-wells plate, I do not use 48 tips for 48 wells, I reuse the tip for transferring media to many wells until the tip seems not sterile anymore then I change the tip. If I need to put down pipette for a little bit, the pipette needs to be put in a way that the tip will not touch anything. If I am not sure that the tip is still clean, it goes to trash as I have stated in the previous comment that ‘always make sure that the tip will not be touched before reuse it’. It does not mean that I clean the used tip and use it again next time.

With 2 contaminations in the past 7 years, I believe I am doing fine with my cell culture and sterile techniques.

Thank you everyone for sharing your experience here and I apologize for the word ‘reuse’ that might lead to misunderstanding.